The combination of PTX and MET inhibited the growth of human umbilical vein endothelial cells. Therefore, it was possible to generate dual-drug-loaded woven vascular stent-grafts to achieve the more effective treatment of AAA.In-stent restenosis caused by tumor ingrowth increases the risk of secondary surgery for patients with abdominal aortic aneurysms (AAA) because conventional vascular stent grafts suffer from mechanical fatigue, thrombosis, and endothelial hyperplasia. For that, we report a woven vascular stent-graft with robust mechanical properties, biocompatibility, and drug delivery functions to inhibit thrombosis and the growth of AAA. Paclitaxel (PTX)/metformin (MET)-loaded silk fibroin (SF) microspheres were self-assembly synthesized by emulsification-precipitation technology and layer-by-layer coated on the surface of a woven stent via electrostatic bonding. The woven vascular stent-graft before and after coating drug-loaded membranes were characterized and analyzed systematically. The results show that small-sized drug-loaded microspheres increased the specific surface area and promoted the dissolution/release of drugs. The stent-grafts with drug-loaded membranes exhibited a slow drug-release profile more for than 70 h and low water permeability at 158.33 ± 17.56 mL/cm Abdominal aortic aneurysm (AAA) is a fatal vascular surgical disease, which causes damage to structures near the aortic wall, and the mortality for patients with ruptured AAA is as high as 85% . VasculaDiabetes medications, such as metformin (MET), can regulate blood sugar and protect the cardiovascular system and have been used to inhibit AAA growth and induce tumor cell apoptosis . MaestreSilk fibroin (SF), derived from the silkworm, is a protein-based biomacromolecule consisting of hydrophobic and hydrophilic blocks . The hydHere, we present a woven vascular stent-graft modified with SF-based paclitaxel/metformin/methanol microspheres (SF-PTX-MET-MT microspheres), which offers a slow-release drug function and inhibits thrombosis and the growth of AAA. SF-PTX-MET-MT microspheres were synthesized by emulsification-precipitation technology and modified on the surface of a woven stent with electrostatic bonding. The surface morphology, thickness, surface hydrophilicity, radial tensile strength, longitudinal tensile strength, water permeability, microstructure, and drug release properties before and after coating were investigated. The safety and effectiveness of the drug in the covered stent were verified by the human umbilical vein endothelial cell line. Degummed silk was purchased from Xiehe Silk Incorporation . Polyethylene glycol  and methanol (MT) were purchased from Sinopharm Chemical Reagent Co., Ltd. . MET was purchased from Ketong Incorporation . PTX was bought from Bailingwei Chemical Technology . Lithium bromide was obtained from Aladdin Industrial Corporation . Polyallylamine hydrochloride (PAH) was obtained from Jiangsu Argon Krypton-Xenon Material Technology Co., Ltd. . Human umbilical vein endothelial cells (HUVECs) were derived from healthy human umbilical vein tissue and purchased from Biotechnology Co., Ltd, Shanghai Enzyme Research Biotechnology Co., Ltd. . They were obtained from the American Tissue Culture Collection (ATCC).v/v/v = 10:10:1); after being incubated in the dark at room temperature for 12 h, deionized water was added to a centrifugal wash by uniformly ultrasonic dispersion, which was repeated three times to obtain drug-loaded SF microspheres. Note that microspheres with different combinations of SF, PTX, MET, and MT were prepared .The stent was woven with PET filaments in plain weave, and the stent-graft was coated with drug-loaded membranes and fabricated, as shown in −1 were analyzed with a Thermo spectroscopy instrument. The X-ray diffraction (XRD) intensity of the lyophilized powders of drug-loaded microspheres was recorded by a PANalytical X-ray diffractometer. The diffraction angle ranged from 5° to 45°, the X-ray wavelength λ was 1.540, and the scanning speed was 0.04°/s. The thermogravimetric analysis (TGA) of drug-loaded microspheres (5 mg) was carried out by purging using a TGA instrument from the TA company, and the heating rate was performed at 15 °C/min.The morphology of SF microspheres and their drug-loaded membranes were observed using a Hitachi scanning electron microscopy (SEM). One hundred drug-loaded microspheres were randomly selected from one SEM image, and the average diameters of these microspheres were analyzed using Image J (1.47 v). The specific surface area of microspheres was estimated from these diameters. Fourier transforms infrared (FTIR) measurements of dried drug-loaded microspheres/membranes from 400 to 4000 cm2, and the measuring pressure was 981 Pa.The thickness, mechanical properties, and water permeability of drug-loaded membranes were tested according to the ISO 7198 2016 standard. The thickness of the vascular stent-grafts with drug-loaded membranes was measured by the fabric thickness gauge (YG(B)141D). For each sample, five different areas were selected for thickness testing, and the average value was recorded. The measuring area was 0.5 cmF (N/mm) is the tensile strength of the drug-loaded membranes; T (N) is the breaking strength of the drug-loaded membranes; D (mm) is the inner diameter of the drug-loaded membranes.Tensile strength in the axial/longitudinal direction of drug-loaded membranes was tested by a material testing machine . The length and width of the samples were 40 × 5 mm to ensure a working distance of 20 mm. The tensile strength was measured as shown in Equation (2).2 area of each sample was randomly selected for contact angle measurement using an optical contact angle measuring instrument (Kruss DSA 100). Water permeability, i.e., the water flow per unit area of the drug-loaded membranes in unit time, was measured under the hydrostatic pressure of 120 mmHg. The membrane is non-porous if the water flow rate is less than 300 mL/min·cm2. Five areas were tested for each stent-graft, and the average contact angle was taken.A 1 cmMET = 0.07744Xabs + 0.39623 (R2 = 0.999) and YPTX = 0.03873Xabs − 0.38474 (R2 = 0.999). The encapsulation rate (ER) and drug loading rate (DR) of MET/PTX in the drug-loaded are given in Equations (3) and (4).The drug loading efficiency of MET and PTX in the microspheres system was determined by establishing an absorbance–concentration standard curve. Therefore, MET/PTX was dissolved in a 9.3 mol/L LiBr solution with seven concentration gradient dilutions, and the absorbance at 231/230 nm of seven concentrations was measured using an ultraviolet spectrophotometer (Cary 5000). The linear regression equations of absorbance with drug concentration were YMET = 0.11389Xabs − 0.1797 (R2 = 0.999) and YPTX = 0.06412Xabs + 0.35086 (R2 = 0.999), respectively. The cumulative release rate of MET/PTX at each time point is as follows:nC is the concentration of the Nth sampling point; V0 is the volume of the release medium; V is the volume of each sampling; W is the total mass of the sample; X is the drug loading rate (%).The 10 mg/mL drug-loaded microspheres/membranes were soaked in phosphate-buffered saline (PBS) solution, which was placed on a shaker in an oven at 37 °C and oscillated at a frequency of 100 times/min. The released solution of 2 mL was collected at 1, 2, 4, 7, 12, 24, 48, and 72 h intervals, and 2 mL of fresh PBS solution was added after sampling. The UV absorbance wavelength of 233 nm and 227 nm was used for MET and PTX, and the concentration of MET and PTX was calculated according to the absorbance-concentration standard equation Y2. The HUVEC were subjected to recovery and progressed down the passage until more than 80% of the cells had aggregated. Punches of sterilized (60Co radiation at 18 kGy) drug-loaded membranes were placed in a 24-well plate and seeded with cells. It can be noted that the same-sized samples of drug-loaded stent-grafts were collected using a hole puncher. Each well was inoculated with 1 × 104 cells. A test group of blank samples was compared to membranes coated with PTX, MET, or a combination of PTX/MET. To calculate cell viability and the inhibition rate of drug-loaded stent-grafts, absorbance values were detected using a Bio-Tek microplate reader with the CCK-8 method. The cell viability and inhibition rate are given by Equations (6) and (7).bX is the absorbance of wells with the culture medium and CCK-8 solution, without the cells and drugs. cX is the absorbance of wells with cells, the culture medium, and a CCK-8 solution. sX is the absorbance of wells with cells, culture medium, the CCK-8 solution, and drugs. HUVECs were stained with calcein for 20 min after being cultured for 48 h, and then the cell growth status was observed using an inverted fluorescence microscope from Caliper life science.HUVEC were cultured as a monolayer in a high-sugar medium at 37 °C and 5% COSF molecules are composed of hydrophilic and hydrophobic fragments. Its secondary structure can be changed under external physical conditions  and chemical stimulations  from random coiling to α-helix or β-sheet ,33. Simi1)). Interestingly, once the drugs were added into the SF microspheres system, their pellet-forming changed. SF-PTX-MT microspheres are rough, porous, and small compared with SF-MT microspheres because PTX is a hydrophobic drug, which has an impact on the hydrophilic and hydrophobic environment of the solution and hinders the self-assembly process of SF molecules. The opposite is that MET is a hydrophilic drug [SF microspheres with and without loading drugs showed excellent pellet-forming properties, uniform sizes, and flat roundness, and there was no adhesion phenomenon in lic drug , which cThe yield of drug-loaded SF microspheres has important an economic reference for the cost control and drug loading of drug preparations. As shown in −1, 1540–1555 cm−1, 1235 cm−1, and 650–670 cm−1, as shown in the peak of Amide I, II, III, and V, respectively. The four peaks of Silk Ⅱ were around 1535–1615 cm−1, 1525–1541 cm−1, 1260 cm−1, and 690–700 cm−1. It can be seen from −1 and indicated the presence of α-helix and random coil structures in pure SF microspheres, which mainly belonged to the Silk Ⅰ structure. The peak at 1625 cm−1 that accompanied a β-sheet structure was found in the SF microspheres with MT, MET, and PTX, indicating that the structure of the drug-loaded SF microspheres changed from an unstable Silk Ⅰ structure to a stable Silk Ⅱ structure. The O-H absorption peak of paclitaxel was 3469 cm−1, and the -NH2 absorption peak of metformin was at 3292 and 3368 cm−1. These results reveal that the drugs were successfully encapsulated in the SF microspheres.Structural analysis can help to understand the formation mechanism of drug-loaded SF microspheres. The absorption characteristic peaks of Silk Ⅰ appear near 1650–1655 cmThe main diffraction angles of Silk Ⅰ were 12.2°, 19.7°, 24.7°, 28.2°, and the angles of Silk Ⅱ were 18.9°, 20.4° . As showThe TGA results of the drug-loaded SF microspheres are shown in The in vitro release of drug-loaded SF microspheres provides information for in vivo behavior after stent implantation. Therefore, the ER, DR, and in vitro release rates were examined. As shown in As shown in Drug-loaded microspheres were clearly observed on the surface of the membrane compared with smooth, untreated PET stents A. Electr2·min) to ensure the non-bleeding performance of artificial blood vessels [p > 0.05), indicating that the modification of the drug-loaded membrane does not affect the thickness of the stent-grafts. The water permeability of the drug-loaded stent-grafts dropped from 163.33 ± 15.28 mL/cm2·min to 158.33 ± 17.56 mL/cm2·min after the modification of microspheres (p > 0.05), which showed that the layer-by-layer self-assembly method of microspheres coating did not greatly affect the water permeability of the stent-grafts.Excellent water permeability is an essential requirement for vascular stent-grafts, which is related to the thickness and morphology of the coated membrane. Generally, the water permeability should not exceed 300 mL/(cm vessels . As showp > 0.05). Since the physical method of attracting positive and negative charges between the microspheres and the coating membrane was used, the microspheres were adsorbed on the surface of the stent-graft without affecting the internal structure of the membrane so the mechanical properties of the coating film could remain stable.The good longitudinal tensile strength provided excellent extensibility of the stent grafts and ensured that there was no major constriction of the stent graft when it opened during surgery by an airbag or by self-expansion . Good raThe good surface hydrophilicity of stent-grafts can help cell growth and coagulation. The water contact angle (WCA) of the uncoated membrane is 112.73 ± 2.04° at the beginning of the measurement, and the WCA decreased dramatically in 20 s, dropping to 2.07 ± 0.87° D. By con−1, which mainly corresponded to the stretching vibration peaks of -CH3 in PTX and MET, indicating that the functional microspheres were attached to the surface of stent-grafts. The absorption peak intensity at 1098 cm−1 and 1247 cm−1 of the stent-grafts was significantly increased compared with the untreated stents, corresponding to the stretching vibration peak of the C-O group bond and the stretching vibration peak of the O-C-O bond, respectively, which further confirmed that the drug-loaded SF microspheres were coated on the surface of stent-grafts.As shown in The in vitro release of stent-grafts with drug-loaded membranes was determined. The drug-loaded membranes exhibited a sudden release within the first 8 h and kept a slow cumulative release rate, which was reached between 8 and 72 h. The cumulative release of MET and PTX reached 51.3% and 21.09%, respectively F. This ip < 0.05). Notably, there was a significant difference in the inhibition of cell growth between PTX and PTX/MET-loaded membranes at 12 h (p < 0.05), suggesting that the addition of MET had a significant inhibitory effect on endothelial cell formation. The dual drug-loaded membranes showed the best cell inhibition rate compared with single MET or PTX-loaded membranes, which were maintained for 60.51 ± 0.05% at 48 h. The characterization of cell viability could also reflect the cell inhibition rate. PTX/MET-loaded membranes showed a significant decrease compared to PTX-loaded membranes at 12 h (p < 0.05) and further confirmed that the addition of dual drugs had an inhibitory ability on cell growth. In contrast to the cell inhibition rate, the cell viability of drug-loaded membranes increased as the cell proliferation time increased (p < 0.05). However, the cell viability of PTX/MET-loaded membranes decreased significantly during this period. The cell viability of PTX/MET-loaded membranes reached the lowest 43.72 ± 0.022% at 48 h compared to single drug-loaded membranes, exhibiting the best cytostatic effect consistent with experiments of cell inhibition.The growth inhibitory effect of the drug-loaded membrane on HUVECs was verified. In addition, the effect of a combined medication was tested. As shown in i)). With the addition of drugs in the membranes, the growth status of endothelial cells in groups of PTX and MET-loaded membranes showed lower cell density and weakened fluorescence intensity. The endothelial cell density of group (i)) showed a significant downward trend, indicating that the PTX/MET-loaded membranes had a strong inhibitory effect on the growth of endothelial cells, consistent with the expected test results. As anti-tumor drugs, PTX and MET play a synergistic role in inhibiting the growth of tumor cells, and the experimental results of the drug-loaded membrane covering inhibiting the growth of endothelial cells provide a theoretical basis for further clinical application of artificial vascular stent-grafts.The HUVECs in the blank group grew in multiple layers and overlapped each other, with clear borders and strong cell viability (Conventional vascular stent grafts for the treatment of AAA often lack the capability of sustained cell growth inhibition. In this work, woven vascular stent-grafts with SF-PTX-MET-MT microspheres modification were developed to inhibit cell growth. This was tested with HUVECs in vitro. Particularly, SF-PTX-MET-MT microspheres were synthesized by emulsification-precipitation technology and layer-by-layer coated on the surface of a woven stent with electrostatic bonding. SF was used as a drug carrier, and the high hydrophilicity of PEG and MT was used to induce the changes in SF molecular structures, resulting in the self-assembly of microspheres. The addition of PTX and MET made the microspheres porous and small. This small structure could increase the specific surface area and promote the release and dissolution of drugs. Dual drug coatings showed good sustained-release phenomena and growth-inhibitory properties of HUVECs. Stable release kinetics for more than 70 h and the cumulative release of MET and PTX reached 51.3%, and 21.09% were observed. We expected the remaining active ingredients to be released slowly as the SF microspheres decomposed, resulting in long-term drug delivery. Therefore, these woven vascular stent grafts with SF-PTX-MET-MT microspheres have the potential to inhibit thrombosis and the growth of AAA, which is expected to lead to the efficient treatment of the diseased tumor and improve clinical applications.


Breast cancer (BRCA) has become the most diagnosed cancer worldwide for female and seriously endanger female health. The epithelial-mesenchymal transition (EMT) process is associated with metastasis and drug resistance in BRCA patients. However, the prognostic value of EMT-related lncRNA in BRCA still needs to be revealed. The aim of this study is to construct an EMT-related lncRNA (ERL) signature with accuracy predictive ability for the prognosis of BRCA patients.RNA-seq expression data and Clinical characteristics obtained from the TCGA (The Cancer Genome Atlas) were used in the study. First, we identified the EMT-related lncRNA by the Pearson correlation analysis. An EMT-related lncRNAs prognostic risk signature was constructed using univariate Cox regression and Lasso-penalized Cox regression analyses. The model’s performance was validated using Kaplan-Meier (KM) survival analysis, ROC curve and C-index. Finally, a nomogram was constructed for clinical practice in evaluating the patients with BRCA and validated by calibration curve and decision curve analysis (DCA). We also evaluated the drug sensitivity of signature lncRNA and the tumor immune cell infiltration in breast cancer.We constructed a 10-lncRNA risk score signature based on the lncRNAs associated with the EMT process. We could assign BRCA patients to the high- and low-risk group according to the median risk score. The prognostic risk signature showed excellent accuracy and demonstrated sufficient independence from other clinical characteristics. The immune cell infiltration analysis showed that the prognostic risk signature was related to the infiltration of the immune cell subtype. Drug sensitivity analysis proved ERLs signature could effectively predict the sensitivity of patients to common chemotherapy drugs in BRCA and provide guidance for chemotherapy drugs for high-risk and low-risk patients.Our ERL signature and nomogram have excellent prognostic value and could become reliable tools for clinical guidance. Breast cancer (BRCA) has surpassed lung cancer to become the most diagnosed cancer worldwide, accounting for more than 30% of estimated new female cancer cases, and it is the second leading cause of female cancer-related death in the United States , 2. EvenEpithelial-mesenchymal transition (EMT), a concept originating from developmental biology, refers to the epithelial cells transdifferentiating into motile mesenchymal cells . Recent that EMT programs do not create mesenchymal cells; instead, they generate migrating cancer cells displaying both epithelial and mesenchymal characteristics . Based oLong non-coding RNAs (lncRNAs) refer to a group of non-coding RNA consisting of more than 200 nucleotides . The effIn the present study, we constructed an EMT-related lncRNA signature prognostic model for BRCA patients and effectively distinguished the risk level of patients. Furthermore, we comprehensively evaluated the tumor immune microenvironment in BRCA patients and analyzed the correlation between EMT-related lncRNA signature and chemosensitivity.We first downloaded the RNA-seq data of 1222 breast cancer samples  with the corresponding clinical characteristic information from the TCGA database. All sequencing data were summarized into the expression matrix of FPKM. The EMT-related gene set consists of 200 genes (https://www.gencodegenes.org/human/) based on the RNA expression matrix annotated by Genome Reference Consortium Human Build 38 (GRCH38) and screened the lncRNAs using the Perl script. Pearson’s correlation analysis using the “cor.test” function in the R package “limma” was then performed between the expression matrix of EMT-related genes and lncRNAs .We first acquired the annotation information of lncRNAs from the GENECODE  are independent prognostic factors in breast cancer patients. Using the R packages “rms” and “regplot”, a nomogram was constructed to predict and visualize the 1-, 3- and 5-year survival rates of breast cancer patients. The Harrell’s concordance index (C-index) could measure the proportion of pairs of patients for which the risk score assigned by the model is in concordance with the actual outcome . A calibPrincipal component analysis is a widespread method for dimensionality reduction and identification of gene distribution patterns, and the R packages “scatterplot3d” were performed to visualize the PCA diagram.The multivariate ROC curve evaluated the predictive accuracy of the risk score and various clinical characteristics on the patient’s survival status, and further assessed the predictive accuracy of each factor by the C-index.We downloaded chemotherapy drug response data from the Genomics of Drug Sensitivity in Cancer (GDSC) database. The R package “pRRophetic” was utilized to predict chemotherapeutic sensitivity to the chemotherapy and targeted drugs recommended in the NCCN guidelines in BRCA patients. The Half-maximal inhibitory concentration , representing the concentration of drug required to inhibit 50% of cell growth, is a valid indicator to assess the sensitivity of breast cancer cells to chemotherapeutic drugs. IC50 differences between high- and low-risk groups were compared using the Wilcoxon test, and p<0.05 was considered statistically significant.We applied Gene Set Enrichment Analysis v 4.1 software for signature lncRNAs pathway enrichment analysis for high- and low-risk groups of BRCA patients. c2.cp.kegg.v7.4.symbol.gmt was downloaded as the reference file, and adjusted p values< 0.05 and FDR< 0.25 were defined as statistically significant.https://cibersort.stanford.edu/) to calculate the relative abundance of 22 immune cells, including B cells, CD4+ T cells, CD8+ T cells, macrophages, dendritic cells, plasma cells, NK cells, mast cells, eosinophils, neutrophils and monocytes in each sample. Only samples with CIBERSORT p<0.05 were retained for further analysis. The infiltration of immune cells and expression of immune checkpoint genes in patients belonging to high- and low-risk groups were analyzed using the Wilcoxon test. The Spearman correlation analysis determined the relationship between immune cell infiltration and the signature risk score. The Tumor Immune Dysfunction and Exclusion (TIDE) algorithm was utilized to predict each patient’s response to immunotherapy.We used the CIBERSORT algorithm (https://lncipedia.org). Unfortunately, we could not find the lncRNA sequences for AC036108.3 and AL390755. Thus, we selected other 8 lncRNAs for in vitro validation. The primers were synthesized by the Tsingke Biotechnology Limited Company . The GAPDH was selected as the internal housekeeping gene, and the relative gene expression was calculated by the 2-ΔΔCt method. Each qRT-PCR assay was repeated three times. We also compared the expression of signature lncRNAs in the subtypes of hormone receptor positive, HER2-amplified and triple-negative breast cancer in the TCGA dataset.The MDA-MB-231 and MCF-7 human breast cancer cell lines and the MCF-10A normal breast epithelial cell line were obtained from Procell . The MCF-10A cell line was cultured in MCF-10A specialized medium CM-0525 . The other cell-lines were cultured in DMEM  supplemented with 10% foetal bovine serum (FBS). RNA was extracted from cells using the TRIzol method with RNA isolater total RNA extraction reagent . A HiScript II QRT SuperMix  kit was used to reverse transcription. qRT-PCR was conducted using the 2X Universal SYBR Green Fast qPCR Mix  kit and the CFX96 Real-time PCR Detection System . We searched the sequences of lncRNAs in the LNCipedia (® Touch Imaging System (Bio-Rad) was applied for the visualization of protein bands.To induce EMT in breast cancer cells, we treated breast cancer MCF7 cells with lactic acid. Lactic acid was purchased from ALADDIN . Lactic acid was diluted to concentrations of 5 mM and 10 mM with medium and treated MCF7 cells for 24 hours. The expression levels of EMT markers E-cadherin and N-cadherin were detected by Western blot. N-cadherin , E-cadherin  and GAPDH  antibodies were purchased from Proteintech . The Bio-Rad ChemiDocThe statistical analysis was conducted by R (version 4.1.2) and Perl (version 5.24) software. A random sequence was generated by SPSS 22.0. Survival curves were evaluated by Kaplan-Meier analysis and log-rank test. p value< 0.05 was considered significantly significant. The Wilcoxon test was used to assess significant differences between the two groups. We plotted ROC curves to evaluate the prediction accuracy of the prognostic signature with area under the ROC curve (AUC) values of 0.6-0.7, 0.7-0.9 and 0.9-1.0 were defined as acceptable, moderate and high accuracy, respectively.To increase the readability of our research for readers, we presented a research flow chart in p value<0.001. Moreover, 32 ERLs associated with BRCA patient prognosis were obtained by univariate Cox regression analysis in the training set (p<0.05). 21 were identified as prognostic good factors (hazard ratio<1), and 11 lncRNAs were identified as bad prognostic factors in BRCA patients (hazard ratio>1). These prognosis-related ERLs are listed in We first used the GENECODE to annotate RNA-seq data in the TCGA-BRCA dataset, and a total of 13162 lncRNAs were annotated. Pearson’s correlation analysis identified 469 ERLs with |correlation coefficient| >0.4 and AC002398.1-0.493*AC036108.3 + 0.311*LINC01705 + 1.280*AC069360.1-1.355*AL390755.1 + 0.818*AC000067.1-0.256*LINC01871 + 0.396*LINC01235-2.335*AC068473.4-1.635*AC104237.3. According to the cut-off point risk score in the training set, patients in the training set  obtained from multivariate Cox regression analysis to quantify the predictors of 1-, 3-, and 5-year prognosis in breast cancer patients , subgroup survival analysis was performed. Except for patients without distant metastasis and HER2 receptor positivity, the ERL signature effectively determined the prognostic risk of patients in the clinical characteristic subgroups (p<0.05)  in entire set of patients and further compared them between patients in the high- and low-risk groups (AC002398.1 was higher in breast epithelial cell lines (AC069360.1, LINC01871 and AC014237.3 was higher in the breast cancer cell lines compared to the breast epithelial cell lines (LINC01705 and AC000067.1 were only significantly highly expressed in the MCF-7 cell line (AC068473.4 was lower in the breast cancer cell lines than in the normal breast epithelial cell line but did not attain statistical significance (The sequences of lncRNAs were presented in the (After lactic acid treatment, the expression of N-cadherin was significantly increased while the expression of E-cadherin was significantly decreased in MCF-7 cells, suggesting that lactic acid significantly induced EMT in breast cancer cell lines (Breast cancer is a malignancy with high incidence and disease burden for females globally, and the identification of novel biomarkers for prognosis and treatment is urgently needed . The epiIn the present study, we constructed a prognostic signature based on the 10 EMT-related lncRNAs. Among these ten lncRNAs, LINC01705, LINC01235, LINC01871 and AL390755.1 have been correlated with breast cancer progression in previous studies. Li et al. found that LINC01705 can serve as a competitive endogenous RNA for sponging miRNA miR-186-5p, regulating TPR expression and further influencing the BRCA progression , 26. LINThe CIBERSORT algorithm was used to assess the relationship between immune cell infiltration and ERLs in the present study. A substantial number of studies have demonstrated that the EMT process is strongly impacted by the tumor immune microenvironment, further promoting the invasion and migration of the tumor cells –31. The Many previous studies have suggested that inhibiting the EMT process in tumor cells may be a method for chemotherapy resistance in breast cancer. Doxorubicin, an anthracycline drug, is widely used in neoadjuvant chemotherapy for BRCA patients, but ERα-negative BRCA patients are less sensitive to doxorubicin than ERα-positive BRCA patients. Ding et al. found that doxorubicin treatment enhances the expression of the crucial EMT transcription factors Snail and Twist via the ERα signal pathway, which promotes the EMT process, resulting in the doxorubicin resistance in the ERα-positive BRCA cells . MicroRNThrough a series of experiments, we proved that AC069360.1, LINC01871, AC014237.3, LINC01705, AC000067.1 and AC068473.4 may be related to the promotion of EMT in breast cancer cells, while AC002398.1 may be related to the inhibition of EMT in breast cancer cells. This is a development first reported by us.in vitro and in vivo experiments. The external validation of the EMT-related lncRNAs signature is lacking in the current study due to the lack of complete lncRNA expression data in the external dataset, such as the GEO and METABRIC. Our ERL signature and nomogram have no examples yet for clinical application, we hope that it will be clinically applicable in the near future. Furthermore, the median follow-up period was less than 3 years, but our future studies will involve longer median follow-up periods to validate these results.However, some limitations in our study should be mentioned: Our research was mainly based on the public database with bioinformatics methods, and whether EMT-related lncRNA effect on the EMT process should be further validated In conclusion, the present study constructed an EMT-related lncRNA signature that predicts the prognosis of BRCA patients, and it also revealed the immune microenvironment and drug sensitivity, providing new insight for breast cancer clinical precision therapy.The original contributions presented in the study are included in the article/in vitro experiments, CL, GX and LZ performed the data analysis. CL, LZ and ZD plotted the figures. CL and LZ wrote the manuscript. YY, QY and XD reviewed and revised the manuscript. All the authors have read and approved the manuscript.CL, JH and GW designed the work, GX and LZ completed


Few studies have investigated the effects of repeated sessions of transcranial direct current stimulation (tDCS) combined with concurrent cognitive training on improving response inhibition, and the findings have been heterogeneous in the limited research. This study investigated the long-lasting and transfer effects of 10 consecutive sessions of multitarget anodal HD-tDCS combined with concurrent cognitive training on improving response inhibition compared with multitarget stimulation or training alone.Ninety-four healthy university students aged 18–25 were randomly assigned to undergo different interventions, including real stimulation combined with stop-signal task (SST) training, real stimulation, sham stimulation combined with SST training, and sham stimulation. Each intervention lasted 20 min daily for 10 consecutive days, and the stimulation protocol targeted right inferior frontal gyrus (rIFG) and pre-supplementary motor area (pre-SMA) simultaneously with a total current intensity of 2.5 mA. Performance on SST and possible transfer effects to Stroop task, attention network test, and N-back task were measured before and 1 day and 1 month after completing the intervention course.The main findings showed that the combined protocol and the stimulation alone significantly reduced stop-signal reaction time (SSRT) in the post-intervention and follow-up tests compared to the pre-intervention test. However, training alone only decreased SSRT in the post-test. The sham control exhibited no changes. Subgroup analysis revealed that the combined protocol and the stimulation alone induced a decrease in the SSRT of the low-performance subgroup at the post-test and follow-up test compared with the pre-test. However, only the combined protocol, but not the stimulation alone, improved the SSRT of the high-performance subgroup. The transfer effects were absent.This study provides supportive evidence for the synergistic effect of the combined protocol, indicating its superiority over the single intervention method. In addition, the long-term after-effects can persist for up to at least 1 month. Our findings also provide insights into the clinical application and strategy for treating response inhibition deficits. Response inhibition comprises the ability to withhold irrelevant or context-inappropriate responses following changes in the environment so that one can make flexible and goal-directed behavioral responses, which is one of the core components of executive function . It is avia anodal stimulation targeting rIFG or pre-SMA , the pre-supplementary motor area (pre-SMA), and the basal ganglia . Transcr pre-SMA .New forms of tDCS emerge as research into the effect of tDCS on enhancing response inhibition progresses. High-definition tDCS (HD-tDCS) is an optimized form of conventional pad-tDCS with high spatial precision and produces more prominent behavioral and neurophysiological effects . MultitaImportantly, repeated sessions of tDCS can increase efficacy through cumulative effects, yield long-lasting after-effects and stable changes in brain function, and are tolerated and safe . Since cHowever, limited studies focused on whether repeated tDCS combined with concurrent behavioral task training further extends response inhibition performance relative to a single intervention method, and the findings are heterogeneous among these few studies. Some studies have shown that this combination can induce greater response inhibition enhancement or better clinical outcomes , with the effects lasting 1 or 2 weeks . HoweverTo fill the research gap, we designed this study to investigate the effects of 10 consecutive sessions of multitarget anodal HD-tDCS targeting rIFG and pre-SMA combined with concurrent cognitive training on improving response inhibition compared with 10 repeated sessions of multitarget stimulation or training alone, including long-lasting effects and transfer effects. Based on available research, we hypothesized that (1) the combined approach would extend and enhance performance improvements of response inhibition compared to multitarget stimulation or cognitive training alone, and the improvement effects would persist to follow-up session , (2) multitarget stimulation or cognitive training alone would induce response inhibition improvements compared to sham tDCS, and (3) the transfer effects would be absent. To the best of our knowledge, this study is the first to examine the effects of repeated daily multitarget anodal HD-tDCS combined with concurrent cognitive training on response inhibition, providing a preliminary insight into strategies to enhance response inhibition ability for both psychiatric and non-psychiatric populations.n = 94, mean age = 20.88 ± 1.77 years, range = 18–25 years, 41 males) had a normal or corrected-to-normal vision, no contraindications to tDCS , and were right-handed as assessed with the Edinburgh Handedness Inventory (n = 24 (stimulation + training group); (2) real stimulation, n = 21 (stimulation group); (3) sham stimulation combined with SST training, n = 24 (sham + training group); and (4) sham stimulation, n = 25 (sham control). Each group underwent intervention separately, without knowing each other. We used G*power 3.1.9.6 to compute a prior sample size with a medium effect size of 0.25, two-tailed α of 0.05, and power (1-β) of 0.80, and a sample of 52 participants was planned (13 per group)  . Writtenvia interviews with the participants after finishing the intervention sessions. All the participants completed the measurements before the intervention (pre-intervention test), the day after the end of the intervention (post-intervention test), and 1 month after intervention (follow-up test). The test contents were identical every time  during the stimulation, and an 11-point scale was used to evaluate the intensity of sensations they felt, ranging from 0 = no sensation to 10 = strongest sensation imaginable , following the procedures for HD-tDCS usage specified in a previous study protocol . The stiaginable .Barratt impulsiveness scale-version 11 was employed to evaluate the impulsivity of the participants. It comprises 30 items and can be divided into three dimensions: attentional impulsivity, motor impulsivity, and non-planning impulsivity, with 10 items in each dimension . In the We used SST to evaluate the response inhibition performance . The tasThe SST was not only the test task for all groups but also the training task for the two groups using SST training. The test SST included a practice block of 48 trials and a formal test block of 200 trials , while the training SST consisted of 48 practice trials and 400 formal trials . The training SST finished within the stimulation duration to guarantee the identical training amount. All the trials were presented at random.The participants performed a classical color-word Stroop task at the pre-test, post-test, and follow-up test, which is a measure of cognitive inhibition . The StrAttentional network test (ANT) is a classic task to study attention ability, which simultaneously measures the efficiency of individual alerting, orienting, and executive control networks involved in attention . The ANTTo probe the far transfer effect on the WM, we used an N-back task that is widely used to measure WM performance . We usedn = 23, 21, 22, 23 for groups 1 to 4, respectively). Five participants were excluded from the Stroop effect analysis due to RT exceeding ± 3 SD of the mean . Concerning ANT, five participants were excluded due to RT deviating >3 SDs of the mean. The final sample for ANT analysis comprised 89 participants . Notably, the number of participants varied by measure because of data filtering of corresponding behavioral measures, which was common practice in previous studies  stop accuracy <0.25 or >0.75 or (2) SSRT <50 ms . After ethe mean . After e studies .post–pre or δfollow–up–pre.We used the IBM SPSS statistical package version 26 to conduct data analyses. The normality in the distribution of data was evaluated using the Shapiro-Wilk test, and the homogeneity of variances was confirmed using Levene’s test. When necessary, the sphericity assumption was verified by Mauchly’s sphericity test, and Greenhouse-Geisser was applied when the sphericity assumption was not met. Categorical variables such as gender and blinding were represented as count or proportion and examined by the chi-squared test. Continuous variables such as accuracy and RT were presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to test baseline performance and continuous data that measured once such as demographic variables. If the outcome measures differed at baseline , they were analyzed by creating contrasts  between the post-test or follow-up test and pre-test to eliminate the interference of baseline, thereby ensuring that any performance changes would be attributable to the intervention. In addition, one-way ANOVA with Bonferroni’s-corrected statistical threshold was used to test group differences of δPost-hoc tests were performed using Bonferroni’s-corrected pairwise comparisons. To further detect the effects of different intervention conditions on improving response inhibition, we conducted a subgroup analysis of SSRT. The participants in each group were separated into high-performance (HP) and low-performance (LP) subgroups based on baseline SSRT via a median-split method (p2) was calculated as measure of effect sizes.Each behavioral task and its outcome measures and BIS-11 scores were tested using a series of 4 × 3 repeated-measures ANOVA (RM-ANOVA) with group  as between-subject factor and time  as within-subject factor. t method . Subgroups > 0.05), including gender distribution, age, years in education, scores of hyperactivity/impulsivity and inattention subscales of ASRS, and sleep duration per night. In addition, one-way ANOVA for scores of BIS-11 and outcome measures of SST, Stroop task, N-back task, and ANT revealed no significant differences in the variables at baseline between the groups (ps > 0.05), except for 2-back accuracy, 3-back accuracy, and orienting effect  = 1.704, p = 0.174, η2p = 0.066]. There were 24 (100%), 19 (90.5%), 23 (95.8%), and 24 (96%) participants in groups 1 to 4, respectively, who believed that they underwent real stimulation. No significant differences were found between the groups in the number of participants reporting real or sham stimulation . The confidence level scores were also non-significant when they were compared between the stimulation + training (8.21 ± 1.29), stimulation (7.57 ± 2.40), sham + training (7.38 ± 2.16), and sham control (8.24 ± 1.76) conditions . The electric field modeling showed that the electric field distribution generated by multitarget HD-tDCS was focused around the anodes and the electric field and current flow produced was largely restricted within the ring of return electrodes  were reported. Most of the participants reported tingling sensation, with 19 (79.2%), 15 (71.4%), 21 (87.5%), and 21 (84.0%) subjects in groups 1 to 4, respectively. Moreover, there was no significant difference in the ratings of the intensity of tingling sensations between the four intervention conditions  (ps < 0.05). Post hoc analysis with a Bonferroni’s-correction showed a significant decrease in SSRT both in the stimulation + training and stimulation alone groups form pre-intervention to post-intervention  and from pre-intervention to follow-up test . It also revealed a significant decrease in SSRT between pre-intervention and post-intervention in the sham + training group (p = 0.037) but not between pre-intervention vs. 1-month follow-up (p = 0.737). Post hoc analysis showed no significant changes in SSRT in the sham control group (ps > 0.999). There were no significant group × time interaction effects for the stop accuracy  and goRT , and the main effects were all non-significant (ps > 0.05).A significant group × time interaction effect on SSRT was observed [= 0.071] . The maiF = 3.538, p = 0.038, η2p = 0.144] and stimulation conditions . The main effects of time and subgroup reached significance in the stimulation + training group (ps < 0.05), and the time main effect was significant in the stimulation group . In the combined intervention (stimulation + training) condition, the Bonferroni’s-corrected post hoc analysis showed significantly decreased SSRT between pre-intervention and follow-up in the HP subgroup (p = 0.002), and between pre-test and post-test (p < 0.001) and between pre-test and follow-up test in the LP subgroup (p < 0.001) (p < 0.001) and follow-up test (p < 0.001) compared to the pre-test in the LP subgroup but not in the HP subgroup . The main effect of the subgroup was significant in the sham + training group . There were no significant main effects in the sham control group (ps > 0.05).Subgroup analysis showed a significant subgroup × time interaction for SSRT in both stimulation + training  due to decreased Stroop effect at the post-test (95.73 ± 5.44 ms) and follow-up test (73.36 ± 4.79 ms) compared to the pre-test (117.28 ± 6.53 ms). The interaction effect of group × time and the main effect of the group were not significant (ps > 0.05). In the ANT, one-way ANOVA showed that both the orienting effect δ values were not significant (ps > 0.05). The time effects for conflict  and alerting  effects were significant but not interaction terms or group effects (ps > 0.05). Concerning the N-back task, the baseline 2-back accuracy significantly differed between the combined intervention and stimulation conditions (p = 0.047). One-way ANOVA showed that the δpost–pre and δfollow–up–pre for 2-back accuracy reached significance , but not 3-back accuracy (ps > 0.05). Post hoc analysis revealed that the combined intervention condition exhibited a smaller 2-back accuracy for δpost–pre (p = 0.022) and δfollow–up–pre (p = 0.013) compared to the stimulation condition. The main effects of time for 2-back RT and 3-back RT were significant (ps < 0.001) due to the reduction of RT at the post-test and follow-up test compared to the pre-test, but the interaction terms and the group effects were not significant (ps > 0.05).In the Stroop task, the main effect of time was significant [nditions , with thps > 0.05).None of the group × time interactions and main effects of time and group for non-planning impulsivity, motor impulsivity, and attentional impulsivity reached significance  and 3-back  accuracy but was not correlated with the Stroop effect in the Stroop task or orienting, conflict, alerting effects in ANT (ps > 0.05).Pearson’s correlation analysis showed that SSRT was significantly and negatively associated with 2-back . Simultaneous activation of shared neural networks by both applied tDCS and performing relevant tasks can produce a synergistic effect. In addition, repeated tDCS and cognitive training may interactively facilitate the beneficial effect which occurs through specific neuroplastic changes such as the N-methyl-D-aspartate (NMDA)-dependent mechanism (Previous studies have proposed that the best effects of tDCS are achieved when the stimulated neural network is already activated or pre-activated (e.g., echanism . The SSTechanism . Based oAdditionally, we found that the repeated sessions of multitarget stimulation or SST training alone could improve response inhibition compared with the sham control condition, consistent with our hypothesis. The favorable effect of multitarget stimulation over sham control on response inhibition is in line with our previous study . It is avia BIS-11 remained relatively stable in our sample that comprised adults aged 18 years and older.We found the good performance of SST was associated with high N-back accuracy at baseline, suggesting a correlation between response inhibition and WM in the mechanism. This is consistent with previous studies that at a behavioral level, response inhibition and WM are correlated , and at Although the 2-back accuracy δ values of the stimulation condition were higher than those of the combined protocol, this was attributed to the baseline difference between the two conditions. Since the 2-back accuracy of the combined protocol was significantly higher than the stimulation condition, it had less room for improvement . TherefoIn this study, to stimulate pre-SMA, we placed central anode at C2. A circuit was formed between the anode and cathodes, which led to current density and electric field existing between the electrodes—between the anode at C2 and the cathodes at Fz and FC4. The detailed simulation –5 showedThe current study has important theoretical and clinical implications. Regarding the theoretical implications, our findings support the synergistic effect of combining tDCS and concurrent cognitive training, indicating better improvement effects than the single intervention method. Moreover, we provided evidence that the combined protocol can be effectively applied in the field of response inhibition enhancement, with the long-lasting after-effects persisting for at least 1 month. Regarding the clinical implication, this study may provide insights into the treatment strategy for the clinical populations with inhibition-deficit-related mental diseases, who need to enhance response inhibition.Despite these important implications, this study has some limitations. First, this study did not use neuroimaging method; therefore, we cannot infer the neural plasticity changes caused by the intervention. In the future, we plan to study brain functional and structural changes induced by this combined protocol. Second, the long-term after-effects were not investigated thoroughly. We only conducted a 1-month follow-up test, and further long-term effects were unknown, which should be dealt with in future studies. Third, this study focused on only young, healthy adults; therefore, it is not known how generalizable our findings are to other populations, such as the clinical sample, and the applicability of our results to other populations requires replication in other samples. Fourth, the study used a single-blinded design due to experimental constraints, possibly weakening the power of this study. Future studies should use more rigorous experimental designs to minimize potential bias, such as the Rosenthal effect. Fifth, the focality of multitarget anodal HD-tDCS in this study has to be improved. The electric field simulation result showed that the maximal electric field strength achieved underneath the anodes C2 and FT8, which we intended to stimulate pre-SMA and rIFG. However, the anode at C2 may also stimulated right motor cortex. Hence, in this study, the electric field produced by the stimulation protocol covered pre-SMA but the precision and focality were not enough, indicating the multitarget stimulation protocol needs to be improved to increase the focality of stimulation. Finally, due to the inter-individual variations of the cortical anatomy and reactivity to stimulation, the individual MRI data should be collected to improve the spatial localization accuracy and the individualized multitarget stimulation protocol for optimal effectiveness is highlighted, and this personalized application might be developed in the future.The present study is the first to use multitarget stimulation combined with concurrent SST training to explore the enhanced improvement effect of response inhibition of this protocol compared to stimulation or training alone. We found that 10 daily sessions of combined interventions and the stimulation alone improved response inhibition, and the effects persisted for 1 month. The training alone only caused improved performance after the intervention. Furthermore, the combined protocol could modulate the performance of the individuals with high baseline response inhibition, which was not seen in the stimulation-alone condition. Notwithstanding the absence of transfer effects, it is too early to conclude that there is no transfer effect, and further studies are warranted. Thus, this study provides supportive evidence for the synergistic effect of the combined protocol. In addition, the long-term after-effect can persist for at least 1 month. Our findings also provide insights into the clinical application and strategy for treating response inhibition deficits.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Tangdu Hospital Ethics Committee, Air Force Medical University. The patients/participants provided their written informed consent to participate in this study.ZG and XZ: concept and design. ZG, RQ, HQ, and HL: acquisition, analysis, and interpretation of data. ZG: drafting of the manuscript. XZ: obtained funding. All authors contributed to the article and approved the submitted version.


The present research aimed to identify the functional effects and underlying mechanisms of metformin on the rat thoracic aorta.-7–10-3 M) when a steady contraction was achieved. The described experimental approach was repeated after incubations with signaling pathway inhibitors and selective blockers of potassium channels to identify the effect mechanisms of metformin. Thoracic aorta segments of Wistar Albino rats were put in the chambers of an isolated tissue bath system. The resting tone was adjusted to 1 g. Following the equilibration time, potassium chloride or phenylephrine was used to contract the vascular segments. The vessel segments were cumulatively treated with metformin (10P<0.001). After the endothelium was removed, the vasorelaxant effect level of metformin was significantly reduced. The level of vasorelaxant effect of metformin was increased by the maintenance of perivascular adipose tissue. Following administrations of L-NAME, methylene blue, compound C, BIM-I, and potassium channel blockers, the level of vasodilatory action of metformin was significantly reduced (P<0.001).Metformin had a potent vasorelaxant effect in a concentration-dependent way (According to the results of this investigation, metformin significantly relaxes the thoracic aorta segments of rats. Metformin-mediated vasorelaxation involves the activation of numerous subtypes of potassium channels, including BKCa, IKCa, Kv, Kir, and K2p channels, as well as endothelium-dependent processes, including AMPK and eNOS/NO/sGS signaling pathways. Moreover, metformin-induced vasorelaxation is mediated through PVAT activation and the PKC signaling pathway.  Diabetes mellitus (DM) is one of the leading causes of morbidity and mortality worldwide. Because of the rise in obesity and the aging population, the incidence of DM is steadily rising. The most common type of DM, which has subtypes including type 1 DM, type 2 DM, and gestational DM, is type 2 DM. DM is a major risk factor for cardiovascular disorders and the main reason for mortality in DM is cardiovascular complications. Metformin (MTF) is an oral antidiabetic drug used as the first stage in the treatment of type 2 DM, even though there are numerous drug alternatives for the treatment of DM nowadays . Galega +) channel subtypes were questioned.Since the 1950s, MTF has been used to manage DM. However, there is very little information available regarding the action mechanisms of MTF. The improvement of endothelial processes is one of the most significant advantages of MTF in the management of DM. This result is crucial in avoiding cardiovascular issues brought on by DM. In this way, the complications and death linked to DM are reduced. Previous studies have highlighted the activation of AMPK (adenosine monophosphate-activated protein kinase) and increased production of nitric oxide (NO) as the mechanisms underlying this effect -10. HoweEthical approval and experimental animalsAfter receiving approval from the Local Animal Experiment Ethics Committee of Bursa Uludag University , the research was launched. In the current research, 20 Wistar Albino male rats  served as experimental subjects. Isolated tissue bath experiments2 · 2H2O, 25 NaHCO3, 1.2 MgSO4 · 7H2O, 1.2 KH2PO4, 11 C6H12O6 · H2O, 4.8 KCl). The surrounding connective tissue and adipose tissue were meticulously removed from the thoracic aorta tissues. Four millimeter vessel segments were prepared after cleaning. Thoracic aortic segments of equal length were placed in the glass bath chambers of the 4-channel isolated tissue bath system . For this procedure, steel hanging equipment and surgical sutures were used. Krebs solution was poured into the bath chambers and reservoirs. A thermal circulator was used to continuously circulate the hot distilled water in the double-jacketed system. The temperature of the Krebs solution in the bath chambers was thus held constant at 37 °C. Krebs solution was continuously gassed with a 95% O2–5% CO2 gas mixture in the bath chambers. As a result, the system’s pH was maintained at 7.4. After the first 30 min, the tone of the vascular segments was adjusted to 1 g. Every 10 min, the Krebs solution which was in contact with the tissues in the chambers was replaced. Throughout the time of equilibration, this process continued (90 min). As a result, the elimination of metabolic wastes and the nutrition of the tissues were both guaranteed. The thoracoabdominal regions of the rats were quickly opened and their thoracic aortas were quickly removed gently. The excised thoracic aorta tissues were placed in Petri dishes containing ice-cold Krebs solution  or potassium chloride  was used to induce vascular contraction following the equilibration interval. When a stable contraction was achieved, cumulative doses of MTF (10-7–10-3 M) were administered to vessel segments. In the control group, distilled water was used in place of MTF in an equivalent volume. To identify the underlying mechanisms by which MTF exhibits its vasorelaxant effects, aorta segments were treated for 30 min with potassium channel blockers or various signaling pathway inhibitors, including N-nitro-L-arginine methyl ester hydrochloride (L-NAME), bisindolylmaleimide I (BIM-I), compound C (CC), indomethacin, and methylene blue. Following that, MTF (10-7–10-3 M) was cumulatively applicated to the vascular segments that had been pre-contracted with PE during the stable contraction of the plateau period (10-6 M).Phenylephrine (10The perivascular adipose tissue preserved (PVAT+) vascular segment samples underwent the same experimental procedure as described above. In this way, the potential contribution of PVAT to MTF-mediated vasorelaxation was examined.-7–10-3 M MTF.To measure the tone changes in the vessel segments, isometric force transducers  were employed. Computer software was used to analyze the resulting changes in vascular tone . The maximum tone value obtained with 10-6 M PE was accepted as 100%. Calculations were made over this value to determine the vascular tone variations in the thoracic aortic segments induced by administration of 10Control group recordings for each experimental group were made at the beginning of the experiments. The same vessel segments were used to finish the experiments in the related experimental groups. The thoracic aortic segments’ equilibration and washing times were repeated for this reason.-6 M) were excluded from the study. Control of endothelial integrity was done by relaxing the vessels that were precontracted with PE with acetylcholine . A relaxation response of more than 80% was thought to be indicative of an intact endothelium (E+) (The vascular segments with the insufficient contractile response obtained with PE (10ium (E+) . -6 M). Less than 10% of the relaxation reaction induced by ACh (10-6 M) indicated that the endothelium was removed. The current study’s experimental methods were all carried out following previous studies’ instructions (Endothelium-denuded (E-) vessel segments were used in some study groups. A wooden toothpick was used to delicately rub the inner surface of the thoracic aortic segments. Then, PE was used to constrict these vessel samples , ACh (10-6 M), MTF (10-7–10-3 M), TEA (10 mM), L-NAME (10-4 M), apamin (100 nM), methylene blue (10-5 M), iberiotoxin (20 nM), BaCl2 (30 µM), 4-AP (1 mM), and TRAM-34 (18 nM). In dimethyl sulfoxide, the following substances were dissolved: CC (10 µM), BIM-I (150 nM), U0126 (1.5 µM), indomethacin (5 µM), glyburide (10 M), and anandamide (10 µM) (DMSO). The ultimate DMSO concentration in the Krebs solution was less than 0.1%. The reactions of vascular smooth muscle (VSM) to contraction or relaxation were unaffected by DMSO.The substances and medications of this research were purchased from Sigma-Aldrich. The literature has established the dosages for MTF, PE, KCl, ACh, L-NAME, methylene blue, BIM-I, CC, indomethacin, and all blockers of the KStatistical analysis-6 M). A one-way ANOVA test followed by Bonferroni post hoc test was used to identify statistical differences between multiple groups.A power analysis was conducted before the research began, and the minimum sample size was determined. The obtained data were statistically analyzed using SPSS v.23.0 . The information was displayed as mean±standard deviation. “n” represents the total number of vessel segments used across all study groups. MTF-induced relaxation responses were expressed as a percentage of the maximal plateau period contraction brought on by PE (10The vasorelaxant effect of MTF in the RTA segmentsP=0.000 at a concentration of 10-6 M). MTF had a significant vascular relaxation effect in RTA (E+). The highest relaxation rate was about 76.23%. (P=0.000 at a concentration of 10-3 M). The highest relaxation rate was around 73.82%  group were found to be significantly lower than the values in the Control (E+) group ( 76.23%. . Additiod 73.82% . These fParticipation of endothelium in the vasorelaxant effect of MTF on the RTA segmentsP=0.000 at a concentration of 10-3 M). Due to the removal of the vascular endothelium, the level of MTF-induced vascular relaxation was greatly decreased, down to about 25.16%  group were found to be significantly lower than the values in the Control (E-) group (t 25.16% . These rParticipation of PVAT in the vasorelaxant effect of MTF on the RTA segmentsP=0.000 at a concentration of 10-3 M). By preserving the vascular PVAT, the vasorelaxant effect of MTF was slightly increased, hitting a level of about 84.96%  (E+) group were observed to be significantly lower than the values in the Control (PVAT+) (E+) group . Due to eNOS inhibition, the value of the vasorelaxant effect of MTF in the RTA was significantly decreased . Due to sGS inhibition, the vasorelaxant effect level of MTF in the RTA was significantly diminished  group were determined to be significantly greater than the values in the Control (MTF) (E+) group . Non-selective COX inhibition did not significantly change the magnitude of the vasodilator effect of MTF  group and the Vehicle (DMSO+MTF) (E+) group   group were found to be significantly greater than those in the Vehicle (DMSO+MTF) (E+) group. Inhibiting AMPK significantly decreased the amount of MTF’s vasodilator effect (–10-3 M) . This fiP=0.040 at a concentration of 10-6; P=0.000 at concentrations of 10-5–10-3 M)  group were found to be significantly greater than those in the Vehicle (DMSO+MTF) (E+) group. PKC inhibition substantially decreased the level of vasodilator effect that MTF had (–10-3 M) . These rP>0.05). Inhibition of MAPK did not affect the vasodilator effect of MTF  group and the Vehicle (DMSO+MTF) (E+) group  group were determined to be significantly higher than the values in the Control (MTF) (E+) group  +MTF (E+) group were determined to be significantly higher than the values in the Control (MTF) (E+) group (P=0.000 at concentrations of 10-5–10-3 M) +MTF (E+) group were determined to be significantly higher than the values in the Control (MTF) (E+) group  +MTF (E+) group were found to be significantly higher than the values in the Control (MTF) (E+) group   +MTF (E+) group were determined to be significantly higher than the values in the Control (MTF) (E+) group (P=0.000 at concentrations of 10-5– 10-3 M) +MTF (E+) group were determined to be significantly higher than the percentage tone values in the Vehicle (DMSO+MTF) (E+) group   . As TEA –10-3 M) . IKCa ch–10-3 M) . KV chan–10-3 M) . Kv7.1-7–10-3 M) . Kir cha 10-3 M) . K2p TAS–10-3 M) . Due to ATP and SKCa channels were selectively blocked by 30 min of glyburide and apamin incubations. There was no significant difference between the tone values in the Apamin (an SKCa channel blocker)+MTF (E+) group and the Control (MTF) (E+) group (P=1.000 at concentrations of 10-7–10-3 M) (ATP channel blocker)+MTF (E+) group and the Vehicle (DMSO+MTF) (E+) group (p=1.000 at concentrations of 10-7–10-3 M)  . Additio–10-3 M) . Followi+ channels have been shown to contribute to MTF-induced vasorelaxation.MTF, which is an effective drug that provides significant benefits in the treatment of DM, still maintains its importance today, as obesity and DM increase as an epidemic. The French doctor Jean Sterne published the first study on the use of MTF in the treatment of DM in 1957 . Its useIn previous studies, the functional effects of MTF administration in various smooth muscle tissues such as the uterus, ileum, and corpus cavernosum have been investigated. Hehir and Morrison  reported-7–10-3 M, significantly caused vasorelaxation in the RTA. The RTA model is proposed as the gold standard model for investigating the mechanisms of action of the drugs which have antihypertensive and vasorelaxant effect potential  and binds to the sGS receptor in the cytoplasm, activating mechanisms mediating vasodilation . The resechanism . Therefoechanism . In thatet al. (10) reported that there was no significant change in PE-induced rat aorta contraction after 30 min of MTF incubation. In contrast, the researchers determined that PE-induced aorta contraction was significantly inhibited after 2 hr of MTF incubation. The researchers who showed that CC incubation inhibited this effect of MTF found an increase in AMPK expression. Therefore, it was concluded that the vasorelaxant effect of MTF was related to AMPK. In the present study, MTF was applied to the contracted RTA with PE and it was determined that it has a relaxing effect. It was shown that the vasorelaxant effect of MTF appeared in a short time. Furthermore, consistent with the study of Pyla et al., CC administration inhibited the vasorelaxant effect of MTF. The maximal vasorelaxant effect level of MTF in this experimental group was determined as approximately 34.32%. Therefore, in addition to its role in both other smooth muscle functions and energy metabolism, the findings indicate that the AMPK signaling pathway is an important mediator in the vasorelaxant effect of MTF in the RTA. Mechanisms such as the stimulation of the eNOS/NO signaling pathway and potassium channel activation after the AMPK signaling pathway may be inducing MTF-mediated vasorelaxation.It is known that the AMPK signaling pathway is associated with VSM contraction and relaxation functions. Vasodilator and hypotensive effects have been reported in various studies using AMPK activators such as AICAR or A769662 , 31. ResNumerous other mechanisms may also be involved in the vasorelaxation effect of MTF, despite studies in the literature that point to AMPK and eNOS/NO signaling pathways as potential mediators. These include mechanisms such as potassium channel activation, as well as PKC and MAPK signaling pathways. The MAPK signaling activation reveals a crucial connection between the contractile and relaxing processes of the heart. According to studies, the porcine palmar lateral vein and the ferret aorta’s contraction and relaxation responses are regulated by an extracellular signal-regulated kinase (ERK) 1/2, which is triggered in the MAPK signaling pathway (36-38). Particularly concerning the impacts of the apelinergic system on cardiovascular contractile functions, the MAPK/ERK 1/2 signaling pathway is a crucial regulator (39). The MAPK/ERK 1/2 signaling and the vasoactive effects of MTF have not been linked in any studies to date. In our research, it was found that the application of the ERK 1/2 inhibitor U0126 did not significantly alter the level of MTF’s vasorelaxant effect. Our findings suggest that the MAPK/ERK 1/2 signaling pathway does not contribute to the vasorelaxant effect of MTF.2+, which leads to increased vascular contraction. eNOS/NO signaling can be activated or inhibited by PKC, regulating VSM contraction and relaxation. PKC is found in a range of vascular beds in various isoforms. The different functional effects of these isoforms are thought to be responsible for PKC’s conflicting effects on vascular functions (et al. (The regulation of smooth muscle contraction and vascular functions may be influenced by mechanisms connected to the PKC signaling pathway, which is involved in numerous cellular processes. PKC can induce vascular contraction by making myofilaments more sensitive to Caunctions . Similar (et al.  recently2+ leads to the contraction of smooth muscle in the arteries. The interaction and creation of cross-bridges between actin and myosin follow the rise in intracellular Ca2+ concentration. A vital regulator of the membrane potential and vascular tone of VSMCs is the physiological activity of different potassium channels, particularly voltage-gated potassium channels. When it comes to controlling how L-type calcium channels open and close, Kv channel activity is a crucial component. Kv channels thus significantly influence the level of cytoplasmic calcium. Voltage-gated Ca2+ channels are inhibited after Kv channels are activated. As a result, VSMCs hyperpolarize, Ca2+ influx decreases and VSM relaxes. However, blocking Kv channels results in the opposite process, which causes VSM to contract (ATP, and BKCa play a role in the vasorelaxant effect of resveratrol. Demirel et al. (ATP, BKCa, IKCa, and K2P channels have a role in the vasorelaxant effect of irisin. In the studies of Sahinturk et al. (ATP, BKCa, and IKCa. In those studies, it was concluded that incubation of potassium channel blockers significantly reduced vascular relaxation caused by peptide agents such as apelin, elabela, and irisin. Those data suggest that potassium channel activation may contribute to the vascular functional effects of MTF, a peptide drug. As a result, in this phase of the research, we looked into the potential contribution of potassium channel subtypes that have been implicated in VSM to MTF-induced vasorelaxation responses. We used both non-selective and selective potassium channel blockers for this reason. Following the application of TEA, iberiotoxin, 4-AP, BaCl2, TRAM-34, anandamide, and XE-991, the vasorelaxant effect level of MTF significantly decreased. On the other hand, the MTF-induced vascular relaxation did not significantly alter following the administration of apamin and glyburide. These findings revealed that the vasorelaxation brought on by MTF involves BKCa, IKCa, Kv, Kir, and K2p channels. However, SKCa and KATP channels are not involved in MTF-mediated vasorelaxant effects. A crucial role for potassium channels in the contraction and relaxation processes of VSM has been suggested by earlier functional studies -16. VSMCcontract , 43. Precontract  determincontract  determinl et al.  determink et al. -15 and ak et al. , it was 2+ response in VSMCs (Our study, along with previous data, suggests that MTF may have beneficial effects on cardiovascular diseases, particularly in the treatment of hypertension, by promoting vasodilation and improving endothelial functions. Several studies in the literature support this claim. For instance, Majithiya and Balaraman  reportedin VSMCs . These fin VSMCs .Ca, IKCa, KV, K2p, and Kir channels. Additionally, endothelium-dependent mechanisms involving AMPK and the eNOS/NO/sGS signaling pathways contribute to MTF-induced vasorelaxation. These findings suggest that MTF may be a promising treatment for hypertensive diseases associated with increased vasoreactivity. The results of this study provide insights for future research on the clinical use of MTF.The present research demonstrates that MTF has vasorelaxant effects on the RTA, and it acts through multiple mechanisms. MTF produces significant vasodilation by activating both endothelium-dependent and -independent mechanisms. The vascular relaxation effect of MTF is further enhanced by PVAT. MTF-induced vasorelaxation is mediated by the PKC signaling pathway and several potassium channel subtypes, including BKAll of this article was prepared and written by Serdar Şahintürk.No financial support has been received. There are no conflicts of interest.


Deep-brain subthalamic nucleus stimulation (DBS-STN) has become a well-established therapeutic option for advanced Parkinson’s disease (PD). While the motor benefits of DBS-STN are widely acknowledged, the neuropsychiatric effects are still being investigated. Beyond its immediate effects on neuronal circuits, emerging research suggests that DBS-STN might also modulate the peripheral inflammation and neuroinflammation. In this work, we assessed the effects of DBS-STN on food-related motivation, food intake pattern, and the level of anxiety and compared them with markers of cellular and immune activation in nigrostriatal and mesolimbic areas in rats with the 6-OHDA model of early PD. To evaluate the potential mechanism of observed effects, we also measured corticosterone concentration in plasma and leukocyte distribution in peripheral blood. We found that DBS-STN applied during neurodegeneration has beneficial effects on food intake pattern and motivation and reduces anxiety. These behavioral effects occur with reduced percentages of IL-6-labeled cells in the ventral tegmental area and substantia nigra pars compacta in the stimulated brain hemisphere. At the same brain structures, the cFos cell activations were confirmed. Simultaneously, the corticosterone plasma concentration was elevated, and the peripheral blood lymphocytes were reduced after DBS-STN. We believe that comprehending the relationship between the effects of DBS-STN on inflammation and its therapeutic results is essential for optimizing DBS therapy in PD. The deep-brain stimulation of the subthalamic nucleus (DBS-STN) is an effective and widely used treatment for motor symptoms in the late stage of Parkinson’s disease (PD) . DBS-STNDBS-STN has been shown to increase cell firing in structures innervated by the STN . MicrodiDissociating the effects of DBS-STN itself from underlying neuropathology and co-occurring pharmacological treatment is critical to understand DBS-STN’s influence on emotion . The objp < 0.001, one-way ANOVA) reduction in the number of TH-positive body cells in the right SNpc of 6-OHDA-depleted rats in both DBS-STN-stimulated (6-OHDA_DBS) and control (6-OHDA_SHAM) animals. The quantification of the number of labeled cells in the SNpc on the lesioned side was reduced by 42%  in 6-OHDA_SHAM and by 35%  in 6-OHDA_DBS (F (3.72) = 128.55) in comparison to the non-lesioned  hemisphere  and TH quantification . The intmisphere C. The Nimisphere D.p ≤ 0.001), DAY_2 , DAY_3 , DAY_5 , DAY_6 , DAY_7 ] (p ≤ 0.001), DAY_2 (H = 6.93 p ≤ 0.01), DAY_3 (H = 7.53 p ≤ 0.01), DAY_4 , DAY_5 , DAY_7 (H = 4.79 p ≤ 0.05), p ≤ 0.01), DAY_2 , DAY_3 , DAY_5 , DAY_6 , DAY_7 , The Kruskal–Wallis test revealed that 6-OHDA_DBS rats have significantly shorter eating times for a single piece of pasta from day 1 to 3 and from day 5 to 7 of the stimulation procedure (DAY_1 to DAY_3 and DAY_5 to DAY_7) in comparison to the 6-OHDA_SHAM group [DAY_1 ] A. The Krp < 0.001) and number of closed-arm entries  and time spent in each of the arms  in comparison to 6-OHDA_SHAM animals. The Dunn test confirmed that all 6-OHDA lesioned rats showed an increased level of anxiety after 6-OHDA injection (DAY_1 and DAY_2) compared to the respective group baseline conditions  and 6-OHDA_DBS animals (p < 0.01 and p < 0.01) were confirmed using Dunn post-hoc tests. At the same points of the procedure, the time spent on open arms was reduced only in 6-OHDA_DBS rats . The anxiolytic-like behaviors were observed in DAY_7 of DBS_STN and were reflected by a higher number of entries and time spent in open arms for 6-OHDA_DBS rats in comparison to 6-OHDA_SHAM rats  and in comparison to DAY_1  = 3.804; p < 0.05; p < 0.05, Tukey post-hoc).The immunofluorescence study showed that the number of IL-6+- and cFos+-containing cells in SNpc and VTA dopaminergic regions in the hemisphere ipsilateral to a lesion were affected by DBS-STN applied in the 6-OHDA-induced model of PD A–F. The p < 0.01; p < 0.05; p < 0.05; The Mann–Whitney U test showed that the plasma corticosterone concentration was elevated after DBS-STN applied during neurodegeneration in the nigrostriatal system in rats  systems as well as activated cFos-labeled cells in these systems were confirmed.The neurotoxin 6-OHDA administered to the SNpc induced neuronal death mainly in the anterior part of the SNpc. During the Vermicelli test, atypical behaviors characteristic of the complete abolition of transmission in the nigrostriatal system were not observed . MoreoveRats with partial 6-OHDA lesions in the nigrostriatal system displayed motivation deficits in an operant task . SNpc-leThe STN is functionally connected with the VTA and hypothalamus . The mecIn addition to the cFos marker, we assessed the percentages of IL-6-labeled cells in both the VTA and SNpc as indicators of neuroinflammation, which arises following the injection of 6-hydroxydopamine into the nigrostriatal system . A distiThe brain cytokine IL-6 supports healthy weight maintenance in a normal physiological state . The perSeveral lines of evidence indicate that corticosterone is an anxiety level modulator . FurtherThe influence of stress hormones on peripheral lymphocyte distributions is well documented . In ourOur study was limited regarding its low number of animals per group due to the very high experimental effort involved in such cohorts. We tried to minimize this limitation by using closely matched groups according to their VHT behavior. It is relatively difficult to train rats in the VHT test towards the constant food intake pattern, so finally, only six rats per group were chosen for further experiments. Given the limited number of rats in each group and the deviation of the results from a normal distribution, our primary approach for statistical analysis involved the use of non-parametric tests such as the Kruskal–Wallis and Mann–Whitney U tests. Both abovementioned tests enable the statistical evaluation of results in the absence of heterogeneity of variance to test an experimental hypothesis . NeverthThe other limitation of our study was that DBS-STN is typically applied in advanced stages of PD to alleviate motor symptoms, and the timeline of our study did not align with this clinical practice. In our study, the 6-OHDA was administered into the SNpc 24 h prior to the first DBS-STN session. In consideration of prior studies, the motor symptoms are conventionally evaluated no earlier than 3 weeks after the neurotoxin administration ,82,83, aThe main effect of DBS-STN obtained in this study has not been shown so far, and we suggest that it may be caused by anti-inflammatory properties of stimulation on the mesolimbic and nigrostriatal systems. In this study, we demonstrated that the application of DBS-STN during neurodegeneration reduced the percentages of IL-6 labeled cells in the VTA and SNpc and increased the percentage of cFos-positive (activated) neurons in the VTA. The peripheral effects of DBS-STN included an increase in corticosterone levels and a decrease in the number of peripheral blood lymphocytes. n = 12), weighing 280–300 g at the time of surgery, were used for the experiments. Animals were kept in standard housing conditions with ad libitum access to standard rat diet  and water. Rats were handled on a daily basis to minimize stress caused during experimental procedures. Animals were split into groups: n = 6),6-OHDA_DBS-group—DBS-STN after intranigral injection of 6-hydroxydopamine (6-OHDA) to mimic PD (n = 6) as control group.6-OHDA_SHAM group—unstimulated rats with intranigral injection of 6-OHDA  using an isoflurane pump , and analgetic butorphanol 2.0 mg/kg i.s.  was administered. A stainless-steel guide cannula  was implanted 1 mm above the SNpc according to the stereotaxic coordinates: −5.3 mm anterior to the bregma, −2.4 mm lateral to the midline, and 6.4 mm below the skull surface  using stDuring the infusion procedure, each animal was held gently by hand. The 6-OHDA  was injected into the right SNpc in a volume of 2 μL (1.5 μg/μL dissolved in 0.9% NaCl containing 0.1% ascorbic acid). Injection was performed using a microinfusion pump  and a Hamilton syringe (10 μL) connected via polyethylene tube to an injection cannula , which was placed into the guide cannula. Injection rate was 0.5 μL/min, and the cannula was left in place for additional 5 min after injection to allow for diffusion into tissue. To protect noradrenergic neurons from damage, animals received an intraperitoneal injection of the noradrenaline reuptake inhibitor desipramine  30 min prior to neurotoxin injection [After a three-week recovery period, rats were habituated to DBS-STN conditions for three consecutive days. Animals were taken from their home cages and placed in transparent plexiglas testing cylinders  and were allowed to explore the boxes for 30 min. DBS-STN was performed on the next day after 6-OHDA injection in accordance with method that we described previously . StimuliThis test was performed immediately before 6-OHDA microinjection (as baseline), one day after 6-OHDA administration, and on 7th day of HFS-STN stimulation (immediately after end of HFS-STN procedure) see . The EPMVHT testing began three weeks after surgery. Procedures for the VHT were based on those developed by Allred et al. . To allog) to obtain fresh plasma without platelets and cells. The supernatant was transferred to Eppendorf tubes, quickly frozen at −70 °C, and stored to analyze plasma CORT. The second part of the sample was tested immediately using automated hematology analyzer (Cell Dyn 3700). Peripheral blood hematological parameters including the total white cell (WBC) number and percentages of lymphocytes (LYM), neutrophils (NEU), and monocytes (MON) were determined in the EDTA_K2 blood as we previously described [Blood samples were collected via heart puncture under 2.5% isoflurane anesthesia (airflow: 0.5 L per min) using an isoflurane pump  between 09.00 and 10.00 AM and 1 h after the DBS-STN or sham stimulation. The blood samples were divided into two tubes. One of tubes was centrifuged 10 min, 3000× 00× g to Plasma corticosterone concentrations were measured via radioimmunoassay using a commercially available kit  and Wizard 1470 gamma counter  in accordance with previously used method ,87,88. TOne hour after the end of DBS-STN or sham stimulation, rats were euthanized with Morbital (2 mL/kg) and transcardially perfused (via the left ventricle) with 200 mL of 0.9% saline followed by 200 mL of 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS).The brains were removed quickly, postfixed, cryoprotected in a 30% sucrose solution in PBS, and then frozen and kept at −70 °C until cryostat sectioning . Coronal 20 µm thick sections containing the VTA and SNpc (5.04 mm posterior to the bregma) were chosen for the double immunofluorescence method for IL-6 and cFos protein detection. For tyrosine hydroxylase and Nissl staining, the coronal 30 μm thick tissue sections were cut at the level of the 1.92 mm anterior to the bregma (according to ) contain2O2)  and allowed to react for 15–20 min. The reaction was controlled and stopped in Tris buffer when the TH-immunoreactive cells turned brown. The tissue sections were placed on slides, air dried, and, after dehydration with ethanol, mounted with DPX .To determine the loss of dopaminergic neurons in the SNpc, we used immunohistochemical staining of tyrosine hydroxylase (TH) as previously described . BrieflImmunofluorescence processing for IL-6 and cFos protein colocalization (IL-6/cFos+ cells) was performed in accordance with the method that we previously described  with somThe counting of labeled cells’ TH+ cell bodies was performed in three sections of the SNpc (−5.04 mm from Bregma). For TH+ cell body counting, a light microscope, Leica DC300 (magnification 10 × 10), and Leica Qwin Software  were used. Then, the mean value of TH+ neurons from each animal was expressed as a percentage of cell loss on the lesioned side compared to non-lesioned side. 2) was chosen randomly from representative sections within each tested brain area. The borders of the brain structures were determined based on the Paxinos and Watson atlas [Immunofluorescence staining sections were viewed at 20 × 10 magnification by an investigator blinded to the assignment of treatment groups. Slices were examined under a Zeiss Axio Scope.A1 fluorescent microscope with camera (AxioCam) and AxioVision Rel.4.8 software. To quantify immunoreactivity, square test area , dehydrated, and finally mounted with DPX . Animals showing a misplaced electrode were not included in the experimental groups presented above.p ≤ 0.05. The statistical evaluation of the mean TH+ cells in the SNpc, cFos+, and IL-6+ were assessed using one-way ANOVA. The main factor was experimental group (DBS or SHAM). Additionally in each group, a division into two brain hemispheres was applied , which resulted in four independent groups of outcomes. The differences in the means were further analyzed with Tukey’s HSD post-hoc. The behavioral data were analyzed using Kruskal–Wallis non-parametric tests and post-hoc Dunn tests. The hematological data and data from plasma corticosterone levels were analyzed using U-Man Whitney tests. All data were expressed as means ± SEM.The statistical analyses were performed using IBM SPSS Statistics 27.0 and the level of significance was set at